DESCRIPTION: Nearly 50% of advanced HER2-positive breast cancer patients succumb to the disease, even after treatment with precision targeted therapies. Some of the most powerful precision therapies, including the landmark drug Herceptin(r) (trastuzumab), target the extracellular domain of the HER2 protein. Yet, new findings suggest that the presence of HER2 protein isoforms that lack the extracellular domain of the full-length HER2 protein (called truncated HER2 isoforms) make tumor cells non-responsive to precision therapies and correlate with drug resistance. Consequently, treatment decisions will be directly informed by the presence of the full-length HER2 protein (trastuzumab) or high levels of truncated isoforms (small molecule kinase inhibitors). Consequently, the ability to directly measure the truncated HER2 isoforms in sparingly available breast biopsy tissues and with single-cell resolution would yield a tremendous advantage for selecting treatment and assessing the potential for drug resistance. Regrettably, existing immunoassay based tools cannot distinguish the forms of HER2 (p185-erbB2, p110-erbB2, p95m-, p95c-, and p95n-erbB2) present in minute tissue biopsies. No antibodies specific to all forms of the HER2 protein exist. A transdisciplinary research program will create and optimize a suite of powerful microanalytical tools to directly quantify the levels and identities of HER2 protein forms and resistance-related signaling proteins in tissues and cells from HER2-positive breast cancer patient biopsies with the Stanford Breast Cancer Tissue Bank. A Protein Panel of drug-resistance related proteins will be assayed (mTOR, PI3K, HER3, IGF-1R, Akt, VEGF) as a pilot study to understand the role of these pathways in drug resistance. After establishing performance in breast cancer cell lines, in Aim 1 the mWB will assay the isoforms in both fresh-frozen and fixed (FFPE) tissue samples. We will assess differences in signaling and expression between leading edge and interior tumor regions. Further, we will compare with patient outcomes and gold-standard IHC classification. In a complementary Aim 2A, the investigators will introduce and optimize a single-cell resolution western blot to assay the HER2 protein isoforms and drug resistance-related signaling proteins but now for thousands of breast cancer cells with single-cell resolution. Cell-to-cell heterogeneity in the Protein Panel will comprise new HER2-positive breast cancer sub-classifications, which will in turn be assessed for correlation to patient outcomes. In Aim 2B, the single-cell tool will be optimized to provide sub-cellular resolution on the cell surface versus nuclear location of full-length HER2 and a truncated isoform, a currently impossible measurement implicated in drug resistance. Anticipated outcomes seek to eliminate a critical bottleneck to improving the well-being of BCa patients. While HER2 isoforms are a focus of this initial effort, numerous other oncoproteins are isoforms (ER, RON, MDM, MYC, BAG-1, PPM1D, and FLIP) and acknowledged contributors to drug resistance and cancer progression. The unique analytical approaches developed will address open questions regarding the role of isoforms in BCa outcomes and therapy resistance in miniscule tissue biospecimens that are otherwise out of analytical reach.